Chen S Y, Charnecki J, Preston J F, Dickson D W
J Nematol. 2000 Mar;32(1):78-84.
Pasteuria penetrans is an endospore-forming bacterial parasite of root-knot nematodes that has potential as a biological control agent. Biochemical investigations of P. penetrans are limited because of difficulty in obtaining large quantities of endospores free of plant debris and contaminating microorganisms. Our objective was to develop a technique for extraction and purification of P. penetrans endospores from root-knot nematodes. Tomato roots infected with Meloidogyne arenaria that was parasitized by P. penetrans were digested with cytolase. The nematode females along with plant debris were washed with a jet stream of water onto an 800-microm-pore sieve nested on a 250-microm-pore sieve. The materials retained on the 250-microm-pore sieve were centrifuged through a 20% sucrose solution. The resulting loose pellet fraction was collected on a 250-microm-pore sieve and then centrifuged through a 47% sucrose solution. Endospore-filled females were handpicked from the 47% sucrose pellicle fraction. Endospores were released by grinding the females with a glass tissue grinder. The endospores were then filtered through a nylon filter with 8-microm openings, collected by centrifugation, and subjected to buoyant density centrifugation in different media. Further purification by buoyant density centrifugation in a linear gradient of sodium diatrizoate resulted in a preparation of endospores free of debris. This additional step may be desirable for the further characterization of components unique to the endospores.
穿刺巴氏杆菌是根结线虫的一种形成内生孢子的细菌寄生虫，具有作为生物防治剂的潜力。由于难以获得大量不含植物碎片和污染微生物的内生孢子，对穿刺巴氏杆菌的生化研究受到限制。我们的目标是开发一种从根结线虫中提取和纯化穿刺巴氏杆菌内生孢子的技术。用溶细胞酶消化被穿刺巴氏杆菌寄生的感染南方根结线虫的番茄根。将线虫雌虫和植物碎片用水流冲到套在250微米孔径筛网上的800微米孔径筛网上。将保留在250微米孔径筛网上的物质通过20%蔗糖溶液进行离心。将得到的松散沉淀部分收集在250微米孔径筛网上，然后通过47%蔗糖溶液进行离心。从47%蔗糖薄膜部分中手工挑选出充满内生孢子的雌虫。通过用玻璃组织研磨器研磨雌虫来释放内生孢子。然后将内生孢子通过孔径为8微米的尼龙滤器过滤，通过离心收集，并在不同介质中进行浮力密度离心。通过在泛影酸钠线性梯度中进行浮力密度离心进一步纯化，得到了不含碎片的内生孢子制剂。这一步骤对于进一步表征内生孢子特有的成分可能是必要的。
